Anti-cancer activity potentiator

ABSTRACT

A pharmaceutical oil-in-water type microemulsion having an action of potentiating the activities of anti-cancer agents, said micro-emulsion comprises fine particles of a vegetable oil or a triglyceride of a medium-chain fatty acid having 8 to 12 carbon atoms containing 0.1 to 10% (w/v) of N-solanesyl-N,N&#39;-bis(3,4-dimethoxybenzyl)ethylenediamine malate of formula (I) below, ##STR1## an aqueous medium, and, 0.05 to 25% (w/v) of a physiologically acceptable phospholipid for dispersing said fine particles in said aqueous medium.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel lipid emulsion having the activity ofpotentiating the anti-cancer activity of an anti-cancer agent, a methodof potentiating the activities of anti-cancer agents by using it withthe lipid emulsion, and to a method of treating cancer occurring in awarm-blooded animal by administering the novel lipid emulsion incombination with anti-cancer agents to the warm-blooded animal.

2. Description of the Prior Art

N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine of thefollowing formula (II) ##STR2## is a known compound.

The compound of formula (II) is described in the patent literature asone of a series of isoprenylamine derivatives having antiviral andantitumoral activities (see, for example, Japanese Laid-Open PatentPublication No. 192339/1982 and its corresponding U.S. Pat. Nos.4645862, 4658063, 4700002 and 4323008).

In recent years various carcinostats and anti-cancer agents againstsolid tumors such as lung cancer, stomach cancer, breast cancer, bladdercancer and testicular tumor or tumors in the hematopoietic organs suchas leukemia and malignant lymphoma have been developed, but no drug hasyet come out which can completely cure or prevent these malignanttumors. For example, cyclophosphamide (CPA), melphalan (MPL), nimustine(ACNU), carboquone (CQ), vincristine (VCR), vinblastine (VLB), vindesine(VDS), bleomycin (BLM), 5-fluorouracil (5-FU), adriamycin (ADM),cisplatin (CDDP), actinomycin D (ACD), methotrexate (MTX), aclarubicin(ACR), toyomycin (TM), neocartinostatin (NCS), and ifosfamide (Ifos)have been used heretofore therapeutically as anti-cancer agents (theparenthesized letters show abbreviated designations) which may sometimesbe used hereinafter). These drugs are used selectively and specificallyin various areas because of their inherent anti-cancer spectra. Forexample, adriamycin (ADM) has a broader anti-cancer spectrum than otherdrugs, and is used against breast cancer, bladder cancer, lung cancer,testicular tumor, malignant lymphoma, and acute leukemia. However, theefficacy of ADM on these diseases is limited, and cancer cells showingresistance to ADM have appeared. A further cumbersome and complexproblem is that other drugs do not show an anti-cancer action on theseADM-resistant cancer cells.

The appearance of drug-resistant tumor cells also becomes a problem withdrugs other than ADM.

Recently, searching for compounds effective on these drug-resistanttumor cells was considered, and it has so far been found that whenN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine itself or itshydrochloride is combined with ADM, the pharmacological efficacy of ADMcan be potentiated, particularly against ADM-resistant tumor cells. Thisfinding was applied for a patent (Japanese Laid-Open Patent PublicationNo. 200913/1986).

The present inventors further made investigations in order to overcomethe problem of the drug-resistance of tumor cells, and determined thatan acid salt, especially a malate, ofN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine of formula (II)above has excellent anti-cancer activity as compared with the compound(II) and it hydrochloride and can potentiate the pharmacologicalefficacy of not only adriamycin but also other anti-cancer agents,particularly the anti-cancer activity of these compounds ondrug-resistant tumor cells, and that this activity has some degree ofspecificity.

The present inventors have further found that when the malate ofcompound (II) is administered in a form incorporated in lipidmicrospheres by applying the drug delivery system (DDS), the lipidmicrospheres are transferred selectively to tumor cells, and theincorporated malate exhibits its effect in situ, and that this offers aneffective therapeutic method.

SUMMARY OF THE INVENTION

This invention relates to a lipid emulsion containingN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malaterepresented by the following formula (I) ##STR3##

More specifically, this invention relates to a lipid emulsion having theaction of potentiating the activities of anti-cancer agents. By usingthe lipid emulsion in combination with anti-cancer agents, theactivities of the anti-cancer agents can be potentiated.

According to this invention, there is provided a pharmaceuticaloil-in-water type micro-emulsion having the action of potentiating theactivities of anti-cancer agents, said emulsion comprising

fine particles of a vegetable oil or a triglyceride of a medium-chainfatty acid having 8 to 12 carbon atoms containing 0.1 to 10% w/v) ofN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate offormula (I) above,

an aqueous medium, and

0.05 to 25% (w/v) of a physiologically acceptable phospholipid fordispersing said fine particles in said aqueous medium.

In a preferred embodiment, the vegetable oil is pharmaceuticallyacceptable soybean oil.

In another preferred embodiment, the physiologically acceptablephospholipid is a purified vegetable oil phospholipid, preferablypurified soybean oil phospholipid.

Preferably, an isotonizing agent is added to the lipid emulsion of theinvention. Examples of the isotonizing agent are glycerol, sugaralcohols, monosaccharides, disaccharides, and amino acids. Thus, in afurther preferred embodiment, there is provided a microemulsionconsisting essentially of

5 to 50% (w/v) of fine particles of a vegetable oil or a triglyceride ofa medium-chain fatty acid having 8 to 12 carbon atoms containing 0.1 to10% (w/v) of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediaminemalate,

0.05 to 25% (w/v) of physiologically acceptable phospholipid,

an isotonizing agent selected from the group consisting of glycerol,sugar alcohols, monosaccharides, disaccharides and amino acids in anamount sufficient to isotonize the emulsion, and

water.

In still another preferred embodiment, there is provided apharmaceutical oil-in-water micro-emulsion consisting essentially of

5 to 30% (w/v) of fine particles of soybean oil having dissolved therein0.3 to 3% (w/v) ofN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate offormula (I),

0.5 to 25% (w/v) of a purified soybean oil phospholipid, and

the remainder being water.

According to a second aspect, the present invention provides a method ofpotentiating the activities of anti-cancer agents by administering theanti-cancer agents in combination with the lipid emulsion of theinvention.

According to a third aspect of the invention, there is provided a methodof effectively treating cancer occurring in a warm-blooded animal, whichcomprises administering an anti-cancer agent in combination with thelipid emulsion of the invention to the animal.

In a preferred embodiment, the lipid emulsion of the invention isintravenously administered.

In another preferred embodiment, the lipid emulsion is administeredwithin 24 hours before the administration of the anti-cancer agent.

In an additional preferred embodiment, the lipid emulsion isadministered for the first time within 24 hours before theadministration of the anti-cancer agent, and thereafter, the lipidemulsion is further administered nearly simultaneously with, or severalhours before, the administration of the anti-cancer agent. If desired,this course of administration is thereafter repeated.

The present invention is unique in that the lipid emulsion preparationexhibits an excellent effect at a low dose of the malate of formula (I),and in that when used in combination with an anti-cancer agent, thelipid emulsion preparation markedly potentiates the activities of theanti-cancer agents.

DETAILED DESCRIPTION OF THE INVENTION

N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine of formula(II), one free base of the malate of formula (I) provided by thisinvention, is a colorless transparent oily substance, but the malate offormula (I) is a colorless to pale yellow crystal having a melting pointof 42° to 44° . The malate can be obtained, for example, by thefollowing method.N-solanesyl-N,N'-bis-(3,4-dimethoxybenzyl)ethylenediamine is dissolvedin a suitable organic solvent, preferably an alcohol such as methanol,ethanol or propanol or an ether such as diethyl ether, tetrahydrofuranor dioxane. A solution of malic acid in an amount of 1.0 to 1.5 molesper mole of the compound of formula (II) is added to the above solution.The mixed solution is heated, and then the solvents are evaporated. Theresidue was washed with an ether solvent, and recrystallized to give thedesired malate.

The malate of formula (I) has been found to be very easy to handle inview of the fact that the compound of formula (II) or its hydrochlorideis a resinous ultraviscous compound. The malate of formula (I) itself ischaracterized not only by having anti-cancer activity but also bymarkedly potentiating the activities of other anti-cancer agents whenused with the other anti-cancer agents. The invention is especiallyexcellent in that when the malate of formula (I) is formed into a lipidemulsion, it is incorporated in lipid microspheres of the emulsion.These lipid microspheres are transferred to tumor cells; and in that insitu, the malate of formula (I) incorporated in the microspherespotentiates the activities of the anti-cancer agents, thus providing aneffective therapeutic effect.

Various anti-cancer agents heretofore used clinically can be potentiatedin activity. Specific examples include adriamycin, actinomycin-D,mitomycin C, bleomycin, 5-fluorouracil, peplomycin and cisplatin. Thelipid emulsion may be said to be unique in that it can exhibit anexcellent effect on tumor cells which show resistance to these drugs.

In the present invention, the lipid emulsion containing the malate offormula (I) is administered in combination with other anti-canceragents. Its dose may vary depending upon the method of administrationand a disease to be treated. Generally, its amount as the malate offormula (I) may be 1.0 mg to 2,000 mg/day. The suitable amount is 4 mgto about 1,000 mg/day. In the administration of the lipid emulsion ofthe invention together with the other anti-cancer agents for the purposeof potentiating the activities of the other anti-cancer agents, the doseof the emulsion of the invention differs depending upon the type of theother anti-cancer agents. Generally, the doses of the other anti-canceragents are those conventionally used, and the dose of the malate offormula (I) is selected from those which potentiate the activities ofthe other anti-cancer agents. In actual administration, the lipidemulsion is in a stable dosage form, and this is unique in view of thefact that the compound (II) itself or its hydrochloride cannot be formedinto a lipid emulsion.

To secure fine particles of 1 micron or below in size which can actuallybe administered as a lipid emulsion, the particles should have a chargewithin the range of +20 mV to +50 mV. It has been found however thatcompound (II) itself or its hydrochloride does not have a particlecharge within this range, but particle aggregation occurs.

Accordingly, to obtain a lipid emulsion ofN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine of formula (II)which exhibits the inherent pharmacological efficacy of the compound(II) must be converted to its malate. The lipid emulsion of thisinvention may be said to be unique in respect of this feature.

The pharmaceutical micro-emulsion of this invention is prepared byintroducing the compound of formula (I) into particles of an oil or fatused in the preparation of ordinary lipid emulsions. For example, it canbe easily prepared by dissolving the compound of formula (I) in fineparticles of the oil or fat, and dispersing the fine particles in waterusing an emulsifier to form an oil-in-water emulsion.

The oil or fat which can be used in preparing the pharmaceuticalmicro-emulsion of this invention includes any pharmaceuticallyacceptable oils and fats which are normally used. Specific examplesinclude vegetable oils such as soybean oil, cottonseed oil, rapeseed oiland safflower oil; triglycerides of medium-chain fatty acids having 8 to12 carbon atoms (such as caprylic acid, capric acid and lauric acid),normally abbreviated as MCT; and mono- or di-glycerides of fatty acidshaving 6 to 18 carbon atoms (such as caproic acid, capric acid, myristicacid, palmitic acid, linoleic acid and stearic acid). They may be usedeither singly or in combination. Among them, vegetable oils and Panacet810 (MCT mixture, a product of Nippon Oils and Fats Co., Ltd.) arepreferably used, and pharmaceutically acceptable soybean oil fitting thestandards of medicines stipulated in Japanese Pharmacopoeia is mostpreferred. The amount of such an oil or fat is not strictly limited, andcan be varied widely depending upon the type or amount of thepharmacologically effective compound of formula (I) and/or the otheringredients. Generally, it is 1 to 50% (w/v), preferably 3 to 30% (w/v),more preferably 5 to 20% (w/v).

Unless otherwise stated, all percentages "% (w/v)" used to denote thecontents or amounts used of the ingredients of the pharmaceuticalmicro-emulsion in the present specification and the appended claims meanparts by weight per 100 parts by volume of the final pharmaceuticalmicro-emulsion.

The emulsifier used to disperse the fine particles of the oil or fatstably in an aqueous medium may be at least one compound selected fromphysiologically acceptable phospholipids and nonionic surface-activeagents, preferably the physiologically acceptable phospholipids.Examples of the physiologically acceptable phospholipids include yolkphospholipid, vegetable oil phosphplipids such as soybean phospholipid,and phosphatidyl choline. Examples of the nonionic surface-active agentsinclude polyoxyalkylene copolymers (for example,polyoxyethylene-polyoxypropylene copolymers having an average molecularweight of 1,000 to 20,000), and hydrogenated castor oil polyoxyalkylenederivatives [such as hydrogenated castor oil polyoxyethylene-(40)-etherand hydrogenated castor oil polyoxyethylene-(20)-ether]. Theseemulsifiers can be used either singly or in combination. Preferably, theemulsifiers used in this invention generally have an HLB of 6 to 15,preferably 10 to 14. Among the above emulsifiers, yolk phospholipid andvegetable phospholipids such as soybean phospholipid are preferred. Thevegetable oil phospholipids, particularly purified soybean phospholipid,are most suitable because they have a better emulsifying power and canform more uniform, finer and more stable phospholipid particles than theyolk phospholipid. Desirably, the vegetable oil phospholipids arepurified to such an extent that the phosphatidyl choline content reachesat least 50% by weight, preferably at least 80% by weight. The soybeanoil phospholipid so purified may have an iodine value of generally 30 to50, preferably about 40.

The emulsifier is used in an amount sufficient to disperse the oil orfat particles containing theN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate in anaqueous medium and maintain them stably in it. Depending upon the typeof the emulsifier, its amount is generally 0.05 to 25% (w/v), preferably0.2 to 6% (w/v), more preferably 0.6 to 2.4% (w/v). On the basis of theoil or fat, the suitable amount of the emulsifier is 6 to 24 parts byweight, especially 6 to 15 parts by weight, per 100 parts by weight ofthe oil or fat.

In the micro-emulsion of the present invention, a moderate amount ofdistilled water or deionized water may be used as the aqueous dispersionmedium. If required, a small amount of a pharmaceutically acceptablewater-miscible organic solvent such as ethanol may be incorporated.

As required, an isotonizing agent and other additives such as anemulsification aid and a stabilizer may further be incorporated in themicro-emulsion of the present invention.

Examples of the isotonizing agent include glycerol, sugar alcohols suchas sorbitol and xylitol; monosaccharides such as glycose and fructose;disaccharides such as maltose; and amino acids such as L-alanine,L-valine and glycine. Of these, glycerol is especially suitable.

The isotonizing agent is added to adjust the osmotic pressure of themicro-emulsion to a value nearly equal to that of a body fluid. Theamount of the isotonizing agent is such that its final concentration inthe micro-emulsion is 0.1 to 0.5 mole/liter, preferably 0.25 to 0.35mole/liter.

Examples of the emulsifying aid that can be incorporated include fattyacids having 10 to 20 carbon atoms (such as stearic acid, palmitic acid,linoleic acid and linolenic acid) and salts thereof (such as sodium andpotassium salts), phosphatidyl ethanolamine, phosphatidyl serine andstearylamine. It may be used generally in an amount of up to 0.4% (w/v),preferably 0.01 to 0.2% (w/v). In particular, the fatty acid or its saltcan be advantageously used in an amount of 0.01 to 0.1% (w/v), andphosphatidyl ethanolamine, phosphatidyl serine and stearylamine may beadvantageously used in an amount of 0.05 to 0.3% (w/v), especially 0.1to 0.2% (w/v).

Cholesterol or tocopherol, for example, may be used as a stabilizer.Conveniently, cholesterol may be used generally in an amount of up to1.2% (w/v), preferably 0.2 to 0.4% (w/v), and tocopherol mayconveniently be used in an amount of up to 2.5% (w/v), preferably 0.2 to0.8% (w/v).

Albumin, its fatty acid amide derivatives, and polysaccharides or theirfatty acid ester derivatives may also be used as the stabilizer. Fromthe standpoint of antigenicity, albumin is desirably one derived from ahuman when preparing a pharmaceutical micro-emulsion for humans. Thefatty acid amide derivatives thereof may, for example, be compoundsobtained by amidating 5 to 40% of the entire amino groups present inalbumin with fatty acids having 14 to 18 carbon atoms (such as palmiticacid and stearic acid). Examples of the polysaccharides include dextran,pullulan and hydroxyethyl starch. The fatty acid ester derivatives ofthese polysaccharides may be compounds obtained by, for example,esterifying 5 to 40% of the entire hydroxyl groups present in thepolysaccharides with fatty acids having 14 to 18 carbon atoms such aspalmitic acid and stearic acid. The stabilizer may be added generally inan amount of 0.02 to 5% (w/v), preferably 0.2 to 2.5% (w/v).

The micro-emulsion of this invention may be prepared by usingemulsifying methods known per se. Ordinary homogenizers may be used asan emulsifying machine. To prepare a stable lipid micro-emulsion, it isconvenient to use two types of homogenizer. Specifically, themicro-emulsion of this invention may be prepared by dissolving aneffective amount of the 4-biphenylylacetic acid ester in the oil or fatsuch as pharmaceutically acceptable soybean oil optionally under heat,adding a predetermined amount of an emulsifier such as refined soybeanphospholipid and as required an isotonizing agent and other additivessuch as an emulsification aid or a stabilizer, stirring the mixtureunder heat to make a uniform mixture, adding water, and treating themixture in a homogenizer to prepare a crude emulsion of the oil-in-watertype, and thereafter, homogenizing the crude emulsion by a pressurizedhomogenizer such as Gaulin high-energy homogenizer. The stabilizer andthe isotonizing agent may be added to the resulting micro-emulsion.

Desirably, the above emulsifying operation is carried out generallyuntil the dispersed oil or fat particles in the resulting emulsion havea mean particle diameter of not more than about 1 micron, preferably notmore than 0.3 micron, more preferably 0.1 to 0.15 micron.

N-Solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate offormula (I) as a pharmacologically active ingredient is convenientlyused so that its concentration generally becomes 0.1 to 10% (w/v),preferably 0.3 to 3% (w/v), more preferably 1 to 3% (w/v).

As required, the micro-emulsion of this invention so prepared may belyophilized. The powder obtained by lyophilization can be converted backto the original micro-emulsion when it is dissolved in water. It shouldbe understood that the term "micro-emulsion", as used in the presentapplication, also denotes such a lyophilized form of the micro-emulsion.

Thus, according to one preferred embodiment of this invention, there isprovided a pharmaceutical oil-in-water type micro-emulsion consistingessentially of

5 to 50% (w/v) of fine particles of a vegetable oil or a triglyceride ofa medium-chain fatty acid having 8 to 12 carbon atoms containing 0.1 to10% (w/v) of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediaminemalate,

0.05 to 25% (w/v) of a physiologically acceptable phospholipid,

an isotonizing agent selected from the group consisting of glycerol,sugar alcohols, monosaccharides, disaccharides and amino acids in anamount sufficient to isotonize the emulsion, and

water.

According to a more preferred embodiment of this invention, there isprovided a pharmaceutical oil-in-water type emulsion consistingessentially of

5 to 30% (w/v) of fine particles of soybean oil having dissolved therein0.3 to 3% (w/v) ofN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate,

0.5 to 25% (w/v) of a purified soybean oil phospholipid,

1 to 5% (w/v) of glycerol, and

the remainder being water.

The lipid emulsion provided by this invention containing the compound offormula (I) is parenterally administered by injection or the like.Preferably, it is administered intravenously. When parenterallyadministered, the lipid emulsion has very good migrability to tumorcells (uptake into the tumor cells), and consequently, can stronglypotentiate the activities of other anti-cancer agents.

In the method of potentiating the activities of other anti-cancer agentsby using the lipid emulsion provided by this invention in combinationwith the other anti-cancer agents and the method of treating canceroccurring in a warm-blooded animal by administering the lipid emulsionof the invention to the warm-blooded animal together with the otheranti-cancer agents, the actual administration schedule of the lipidemulsion of the invention is preferably by the following specificmethod.

In one embodiment, the lipid emulsion of the invention is administeredtogether with the other anti-cancer agents. In another embodiment, thelipid emulsion of this invention is administered for the first timewithin 24 hours before the administration of anti-cancer agents, andthen the lipid emulsion of the invention is further administeredsimultaneously with, or several hours before, the administration of theanti-cancer agents, and this administration course is continuously orintermittently repeated. By this administering method, the activities ofthe other anti-cancer agents used in combination are markedlypotentiated, and the therapeutic effect can be effectively exhibited.

Accordingly, the lipid emulsion of this invention contributes greatly tocancer therapy.

The pharmacological activities ofN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine and a lipidemulsion containing the malate will now be illustrated specifically.

[A] PHARMACOLOGICAL TESTS A-I: Anti-cancer activity and potentiationactivity in vitro

Part 1

Procedure

Sensitive cell line V79/S and ADM-resistant cell line V79/ADM of theChinese hamster were used, and sensitive cell line PLC/S andcholchicin-resistant cell line PLC/Col, as human hepatoma cells.

The sensitive cell lines (400 cells) or the resistant cell lines (600cells) were each incubated for 24 hours in 10 ml of Eagle's MEM mediumcontaining 10% bovine serum. Each of the drugs indicated in Table 1below was dissolved in ethanol, and 50 microliters of the resultingsolution was added to the medium. The incubation was further carried outfor 9 days. The concentration of the drug which inhibits 50% of thecolony formation (proliferation) of the tumor cells was calculated.

V79/S and V79/ADM were tested by using all of the drugs described above,but PLC/S and PLC/Col were tested by using only the malate of formula(I), adriamycin and peplomycin.

Results

The concentrations (micrograms/ml) required to inhibit 50% of theproliferation of the tumor cells, expressed by IC₅₀, are shown in Table1.

                  TABLE 1                                                         ______________________________________                                                Cell line                                                                     IC.sub.50 (μg/ml)                                                  Drug      V79/S    V79/ADM    PLC/S  PLC/Col                                  ______________________________________                                        Compound (I)                                                                            11.5     3.0        6.8    1.7                                      ADM       0.057    8.8        0.005  0.08                                     ACR       0.037    0.27       --     --                                       ACD       0.017    0.28       --     --                                       CDDP      0.37     0.32       --     --                                       MTX       0.017    0.0078     --     --                                       Mitoxantrone                                                                            0.019    0.36       --     --                                       PEP       1.95     0.48       0.24   0.19                                     ______________________________________                                    

It is clear from the results given in Table 1 that the malate of formula(I) itself had some degree of tumor growth inhibition activity(anti-cancer activity), and inhibited proliferation of ADM-resistantChinese hamster cells (V79/ADM) or colchicin-resistant human hepatomacells (PLC/Col) in concentrations required to inhibit growth of theirsensitive cell lines; and therefore that the malate of formula (I)showed clear tumor growth inhibition (anti-cancer) activity ondrug-resistant tumor cells and drug-resistance overcoming activity.

In contrast, the adriamycin (ADM) clinically used showed a markedreduction in its anti-cancer activity on ADM-resistant tumor cells. ACRand ACD which are other carcinostats or anti-cancer agents, andmitoxantrone recently marketed for their appreciable anti-canceractivity on acute leukemia, acute lymphoma and breast cancer have largerIC₅₀ values on the proliferation of drug-resistant tumor cells V79/ADMthan those on the sensitive cell lines. Hence, the resistances to thesedrugs were not overcome.

It will be understood that the malate of formula (I) is excellent.

Part 2

Procedure

Lung adenocarcinoma, lung squamous cell carcinoma, osteosarcoma andovarian carcinoma were selected as clinically isolated human cancercells, and the anti-tumor activities of various drugs were examined.Specifically, various tumor tissues were individually cut to thin piecesby a razor, and by washing in saline by using a 1 mm mesh, theconnective tissues were removed. The resulting solution was centrifugedat 1000 rpm for 1 minute to remove necrotizing substances. Washing withsaline gave tumor cells.

The tumor cells obtained as above (about 10 g) were put in 0.4 ml of aculture solution (RPMI 1640 plus 10% calf serum), and each of the drugsin the concentration shown in Table 2 was added. They were incubated at37° for 4 to 8 hours. Thereafter, pumping was carried out to dispersethe tissue pieces. Then, centrifugation at 1000 rpm for 5 minutes, cellcoating and Giemsa staining were performed. The anti-tumor activity ofeach drug was examined on the basis of observation of the morphologicalchanges of the tumor cells under a microscope.

The experiment was conducted on two examples of each tumor cells (total8 examples). The malate of formula (I) was used in a concentration of 3micrograms/ml for 3 examples out of 8, and 30 micrograms/ml on 5examples.

Results

The anti-tumor activities on the tumor cells were examined by using theeffective dose of the drugs in vivo. The results with those exampleswhich were rated as "significant" by the Fisher's significance test aregiven in Table 2.

                  TABLE 2                                                         ______________________________________                                                       Drug con-                                                                     centration                                                                              Efficacy ratio                                       Drug           (μg/ml)                                                                              (*)                                                  ______________________________________                                        Compound (I)   3 or 30   5/8                                                  CDDP           0.5       4/8                                                  CPM            10        3/8                                                  ADM            0.3       3/8                                                  ACNU           2.0       3/8                                                  VLB            0.1       3/8                                                  BLM            1.0       2/8                                                  VCR            0.1       2/8                                                  ACR            0.2       2/8                                                  MPL            0.5       1/8                                                  CQ             0.1       1/8                                                  5-FU           10        1/8                                                  MTX            3.0       1/8                                                  MMC            0.2       0/8                                                  VDS            0.5       0/8                                                  ACD            0.01      0/8                                                  ______________________________________                                         (*)Rated as "significant" by the Fisher's significance test.             

As is clear from the examples given in Table 2, the malate of formula(I) in accordance with this invention was found to have anti-tumoractivity on various tumor cells.

A-II: Anti-cancer activity and potentiation activity in vivo

Part I

Procedure

Three to eight male BDF₁ mice having a body weight of 20±3 g were used,and B16 melanoma cells were selected as tumor cells. The melanoma cellswere homogenized, and the homogenate was transplanted subcutaneously tothe mice in an amount of 0.5 ml/mouse. After 7 days, the malate offormula (I) in accordance with this invention and an anti-cancer agent,neocartinostatin (NCS), were intraperitoneally administered eithersingly or in combination three times every 4 days in a dose of 5 mg/kgand 0.45 mg/kg for each time. The life prolonging rate of the mice wasdetermined by the treated group/non-treated control group (T/C %).

Results

The T/C (%) values are tabulated in Table 3.

                  TABLE 3                                                         ______________________________________                                        Drug              T/C (%)*                                                    ______________________________________                                        Control           100                                                         Compound (I)      165                                                         NCS               135                                                         Compound (I) + NCS                                                                              129                                                         ______________________________________                                         *Comparison at M.S.T. (medium survival time)                             

As is clear from the results given in Table 3, the group to which themalate of formula (I) was administered showed a particularly good lifeprolongation effect as compared with the non-treated control group, andwas also found to have a significant effect over the group to which NCSalone was administered. The group to which these drugs were administeredin combination showed no difference in life prolongation rate ascompared with the groups to which the drugs were administered alone. Inthis test, anti-cancer activity potentiating action was not clearlyseen.

Part 2

Procedure

Male BDF₁ mice having a body weight of 20±2 g (6 per group) were used,and as tumor cells, Lewis lung cancer cells were transplanted under theskin of the side abdomen of each of the mice at a rate of 10⁵ cells.Seven days later, a lipid emulsion containing the malate of formula (I)(the lipid emulsion of Example 2) and ADM as an anti-cancer agent wereintravenously administered in the doses shown in Table 4 below. The lifeprolongation rate of the mice in each group was determined by thetreated group/non-treated control group (T/C %).

Results

The T/C (%) values are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                        Dose                                                          Drug            (mg/kg)  T/C (%).sup.(1)                                      ______________________________________                                        Control                  100                                                  Lipid emulsion.sup.(2)                                                                        2.5      205                                                  of Example 2    5.0      179                                                                  10       103                                                                  20       203                                                  ADM             1.25     218                                                                  2.5      173                                                                  5.0      241                                                  ______________________________________                                         .sup.(1) Comparison at M.S.I.                                                 .sup.(2) Calculated as the malate of formula (I)                         

It is seen from the results shown in Table 4 that the lipid emulsioncontaining the malate of formula (I) shows a marked life prolongationeffect and excellent anti-cancer activity.

Procedure

Male BDF₁ mice having a body weight of 20±2 g, 6 per group, were used.P388 mouse leukemia cells (one week after transplantation) as tumorcells were washed with 0.9% saline and suspended in saline so that thenumber of the cells reached 5×10⁶ /m. The suspension wasintraperitoneally administered to the mice at a rate of 0.2 ml (10⁶cells) per mouse.

Twenty-four hours after the transplantation of tumor cells, the malateof formula (I) and cyclophosphamide (COPA) were successivelyadministered intraperitoneally once to the mice, and the lifeprolongation rate of the mice in each group was determined.

The malate of formula (I) was used after suspending it and Tween 80 at aratio of 1:5 to saline, and this suspension was diluted with a solutionof Tween 80 in the same concentration. CPA, on the other hand, was usedas a solution in saline.

CPA (50 mg/kg and 100 mg/kg) and the malate of formula (I) (10 mg/kg and20 mg/kg) were administered singly or in combination, and the lifeprolongation rate was determined. The life prolongation rate shows therate of the treated group to the control group into which P288 mouseleukemia cells were transplanted (T/C %), and the rate of the group towhich CPA and the malate were administered to the group to which CPAalone was administered (T/C %). The results are summarized in Table 5.The number of surviving mice determined 30 days later was also shown inTable 5.

                  TABLE 5                                                         ______________________________________                                                                         Number of.sup.(3)                            Drug                    T/C.sup.(2)                                                                            surviving                                    (dose)       MST.sup.(1)                                                                              (%)      animals                                      ______________________________________                                        Control group having                                                                       10.1       100      0/6                                          P388 cells trans-                                                                           [9-11]    --                                                    planted therein                                                               CPA (50 mg/kg)                                                                             20.3       201      0/6                                                       [19-22]    (100)                                                 + Compound (I)                                                                 (5 mg/kg)   21.3       211      0/6                                                       [19-29]    (105)                                                 (10 mg/kg)   22.0       218      0/6                                                       [20-23]    (108)                                                 (20 mg/kg)   22.3       221      0/6                                                       [19-23]    (110)                                                 (40 mg/kg)    9.3        92      0/6                                                        [8-26]     (46)                                                 CPA (100 mg/kg)                                                                            24.0       238      1/6                                                       [21-30]    (100)                                                 + Compound (I)                                                                 (5 mg/kg)   29.0       287      0/6                                                       [22-30]    (121)                                                 (10 mg/kg)   >31.0      >341     5/6                                                        [27->30]  (>143)                                                (20 mg/kg)   12.0       119      1/6                                                        [4-30]     (50)                                                 (40 mg/kg)    9.3        92      0/6                                                        [3-11]     (39)                                                 ______________________________________                                         .sup.(1) MST: Medium Survival Time                                            .sup.(2) The parenthesized figures show percentages based on the group to     which CPA alone was administered.                                             .sup.(3) Number of animals which survived for 30 days.                   

The above results show that the malate of formula (I) in accordance withthis invention at low concentrations markedly potentiated theanti-cancer activity of CPA, and particularly in the group to which CPAwas administered in a dose of 100 mg/kg, the combined use of 10 mg/kg ofthe malate exhibited an excellent potentiating efficacy.

A-III Anti-cancer activity potentiating action

Procedure

The anti-cancer activity potentiating action of the malate of formula(I) was examined using Chinese hamster cells (sensitive cell line V79/Sand ADM-resistant cell line V79/ADM), human hepatoma cells (sensitivecell line PLC/S and colchicin-resistant cell line PLC/Col) and mouseleukemia cells (sensitive cell line L5178Y/S) and aclarubicin-resistantcell line L5178Y/ACR).

Of these tumor cells, the sensitive cells (400 cells) and the resistantcells (600 cells) were each incubated in 10 ml of Eagle's MEM mediumcontaining 10% bovine serum for 24 hours, and then each of the testdrugs indicated in Table 6 was added. Thereafter, the incubation wascarried out for 9 days. The concentration (IC₅₀ : micrograms/ml) of thetest drug required to inhibit 50% of the colony formation(proliferation) of the tumor cells was calculated.

Specifically, the experiment was carried out by using the followinganti-cancer agents.

(A) Chinese hamster V79 cells

As anti-cancer agents, there were used adriamycin (ADM), aclarubicin(ACR), actinomycin D (ACD), 5-fluorouracil (5-FU), cytarabin (Ara-C),cisplatin (CDDP), methotrexate (MTX), mitomycin C (MMC), nimustine(ACNU), mitoxantrone, etoposide (VP-10), vincristine (VCR), bleomycin(BLM) and peplomycin (PEP). For the sensitive cells, the anti-canceragents were used in combination with 10 micrograms/ml of the malate offormula (I), and for the resistant cells, the anti-cancer agents wereused in combination with the malate of formula (I) 3 micrograms/ml, 5micrograms/ml, and 10 micrograms/ ml.

(B) Human hepatoma cells

As anti-cancer agents, adriamycin (ADM) and peplomycin (PEP) were usedin combination with the malate of formula (I) (5 micrograms/ml).

(C) Mouse leukemia cells

Adriamycin (ADM) and aclarubicin (ACR) as anti-cancer agents were usedin combination with 20 micrograms/ml of the malate of formula (I).

Results

The results are summarized in Tables 6 and 7.

                                      TABLE 6                                     __________________________________________________________________________            Cell line                                                                     V79/S (parent cells)                                                               Compound                                                                      [in com-  V79/ADM (resistant cells)                                      Anti-                                                                              bination                                                                            Poten-                                                                            Anti-               Poten-                             Anti-   cancer                                                                             with com-                                                                           tiation                                                                           cancer                                                                             Combination with                                                                             tiation                            cancer  agent                                                                              pound (I)]                                                                          activity                                                                          agent                                                                              compound (I)   activity                           agent   alone                                                                              10 μg/ml                                                                         (times)                                                                           alone                                                                              3 μg/ml                                                                         5 μg/ml                                                                         10 μg/ml                                                                        (times)                            __________________________________________________________________________    Compound (I)                                                                          11.5 --    --  3.0  --   --   --   --                                 ADM     0.057                                                                              0.008 7.1 8.8  --   --   0.25 35.2                               ACR     0.037                                                                              0.027 1.37                                                                              0.27 --   --   0.024                                                                              11.3                               ACD     0.017                                                                              0.0032                                                                              5.3 0.28 --   --   0.0063                                                                             44.4                               5-FU    9.7* 2.1*  4.6 7.2* --   0.62*                                                                              --   11.6                               Ara-C   0.076*                                                                             0.054*                                                                              1.4 0.22*                                                                              --   0.035*                                                                             --   6.3                                CDDP    0.37 0.27  1.4 0.32 --   0.13 --   2.5                                MTX     0.014                                                                              0.017 0.8 0.0078                                                                             --   <0.0025                                                                            --   >3.12                              MMC     0.02 0.01  2.0 0.057                                                                              --   0.011                                                                              --   5.20                               ACNU    7.60 5.40  1.4 10.0 --   3.10 --   3.20                               Mitoxantrone                                                                          0.019                                                                              0.0081                                                                              2.3 0.36 0.074                                                                              --   --   4.9                                Etoposide                                                                             0.27 0.098 2.8 >20.0                                                                              7.4  --   --   2.7                                VCR     0.0271                                                                             0.00325                                                                             8.3 0.730                                                                              0.0351                                                                             --   --   20.8                               BLM     2.1  0.01  202 0.56 0.0385                                                                             --   --   14.5                               PEP     1.95 0.0165                                                                              118 0.48 0.0157                                                                             --   --   30.5                               __________________________________________________________________________     *μM                                                                        (IC.sub.50 : μg/ml)                                                   

                                      TABLE 7                                     __________________________________________________________________________            Cell line                                                                     PLC/S (parent cells)                                                                           PLC/Col (resistant cells)                                    Anti-        Poten-                                                                            Anti-        Poten-                                  Anti-   cancer       tiation                                                                           cancer       tiation                                 cancer  agent                                                                              Compound (I)                                                                          activity                                                                          agent                                                                              Compound (I)                                                                          activity                                agent   alone                                                                              5 μg/ml                                                                            (times)                                                                           alone                                                                              5 μg/ml                                                                            (times)                                 __________________________________________________________________________    Compound (I)                                                                          6.8  --      --  1.7  --      --                                      ADM     0.0050                                                                             0.0053  1.0 0.08 0.027   3.0                                     PEP     0.24 0.08    3.0 0.19 0.03    6.3                                     __________________________________________________________________________            Cell line                                                                     PLC/S (parent cells)                                                                           PLC/ACR (resistant cells)                                    Anti-        Poten-                                                                            Anti-        Poten-                                  Anti-   cancer       tiation                                                                           cancer       tiation                                 cancer  agent                                                                              Compound (I)                                                                          activity                                                                          agent                                                                              Compound (I)                                                                          activity                                agent   alone                                                                              20 μg/ml                                                                           (times)                                                                           alone                                                                              20 μg/ml                                                                           (times)                                 __________________________________________________________________________    ADM     0.048                                                                              0.016   3.0 0.06 0.18    3.7                                     ACR     0.019                                                                              0.017   1.0 0.07 0.043   1.8                                     __________________________________________________________________________     (IC.sub.50 : g/ml)                                                       

The foregoing results lead to the discovery that the malate of formula(I) well potentiates the activities of other anti-cancer agents as aresult of combined use. Furthermore, the potentiation activity isexcellent on tumor cells having resistance to the other anti-canceragents.

A-IV: Tumor proliferation inhibiting activity

Procedure

Female BALB/c rats (7 weeks old), 8 per group, were used. Tumor cells(Meth-A) induced by methyl cholanthrene were transplantedintraperitoneally to the rats, and then the malate of formula (I) wasadministered intraperitoneally to the rats. The proliferation inhibitingactivity of the malate was examined by comparing the weight of the tumorcells with that of the tumor cells in the non-treated control group.

The dose of the malate of formula (I) was 1.25, 2.5, 5.0, and 10.0mg/kg/day. The administration schedule was as shown in Table 8 below.Mitomycin C (MMC) was administered as another anti-cancer agent. Theweight of the tumor cells was measured, and the proliferation inhibitingactivity was determined.

Results

The weight (g) of the tumor with respect to that of the tumor in thecontrol group (T/C, %) after 16 days from the transplantation of thetumor cells, and the number of surviving rats after 16 days are shown inTable 8.

                  TABLE 8                                                         ______________________________________                                                                            Number of                                                                     surviving                                           Dosing   Tumor weight     animals                                   Drug.sup.(1)                                                                            schedule mean ± S.D.                                                                            T/C  (16 days                                  (dose)    (day)    (g)         (%)  later)                                    ______________________________________                                        Control   --       1.672 ± 0.530                                                                          100  8/8                                       Compound (I)                                                                   (1.25)   1, 3, 5  1.255 ± 0.533                                                                          75   8/8                                        (2.5)    1, 3, 5  1.327 ± 0.350                                                                          79   8/8                                        (5.0)    1        1.389 ± 0.644                                                                          83   8/8                                       (10.0)    1        1.091 ± 0.659                                                                          65   8/8                                       MMC (1.0) 1-5      0.347 ± 0.185                                                                          21   8/8                                       ______________________________________                                         .sup.(1) Dose in mg/kg/day                                               

The above results show that the malate of formula (I) significantlyinhibited the proliferation Meth-A sarcoma cells in rats.

A-V: Anti-cancer activity potentiating action in vitro

Procedure

ICR nude mice (4-5 weeks old; 7 per group) were used. Human stomachcancer cell line H-81 was used as tumor cells. Tissue pieces, 2 mm insquare, were taken, and transplanted subcutaneously to the right backportion of the nude mice. The volume of the tumor after transplantationwas calculated by long diameter x short diameter x thickness x 1/2.Administration of a drug was started on the 11th day after thetransplantation when the above tumor volume reached about 100 mm³.

The sequence of the drug administration was as follows: A lipid emulsionof the malate of formula (I) (the lipid emulsion of Example 2) wasintravenously administered, and 20±5 minutes later, adriamycin (ADM) wassimultaneously administered intravenously. The administration wasfurther repeated every five days from the day on which theadministration was first carried out. The total number ofadministrations of each drug was four. The doses of the drugs in theindividual groups are as shown in Table 9 below.

Results

The tumor volume of each mouse on the day of measurement was calculatedby the long diameter x short diameter x thickness x 1/2, and the tumorproliferation inhibiting ratio to the control group to which no drug wasadministered was determined. The measurement was made on the days onwhich the drugs were administered, and four weeks after the start ofdrug administration. The results are shown in Table 9.

                                      TABLE 9                                     __________________________________________________________________________                  Tumor growth inhibition rate (%)                                              1st 2nd  3rd 4th  --                                                    Dose  Start-                                                                            5 days                                                                             10 days                                                                           15 days                                                                            4 weeks                                       Drug    (mg/kg)                                                                             ing day                                                                           later                                                                              later                                                                             later                                                                              later                                         __________________________________________________________________________    Lipid emulsion                                                                        10.sup.(1)                                                                          --  15.7 18.9                                                                              17.1 14.4                                          +        2    --  16.5 45.7                                                                              49.5 45.2**                                        ADM      5    --  --   --  --   59.7*                                         Lipid emulsion                                                                         5.sup.(1)                                                                          --                                                              +                 23.5 46.3                                                                              53.1 46.3**                                        ADM      2    --                                                              Lipid emulsion                                                                        10.sup.(1)                                                                          --                                                              +                 24.3 49.9                                                                              57.6 53.3***                                       ADM      2    --                                                              Lipid emulsion                                                                         5.sup.(1)                                                                          --                                                              +                 33.3 83.4                                                                              88.0 84.5***                                       ADM      5    --                                                              __________________________________________________________________________     *p < 0.5,                                                                     **p < 0.1,                                                                    ***p < 0.01                                                                   .sup.(1) Calculated as the malate of formula (I)                              Note 1: With regard to ADM (5 mg/kg), the administration was carried          singly, and the evaluation was made four weeks later.                         Note 2: Assay was carried out on the significance from the control group      only with regard to the results obtained four weeks later.               

It is seen from the results given in Table 9 that the lipid emulsioncontaining the malate of formula (I), when used in combination with ADM,synergistically potentiates the activity of ADM. Particularly, the tumorvolume showed a clear tendency to diminish in the group to which 5 mg/kgof the malate of formula (I) and 5 mg/kg of ADM were administeredtogether. In any of the groups tested, no case of death was noted in thenude mice. Side-effects were neither observed.

It will be understood in view of the foregoing that the lipid emulsionof this invention has a marked anti-cancer activity potentiating action.

A-VI: Anti-cancer activity potentiating action in vivo

Procedure

ICR nude mice, 6 per group, were used, and the experiment was conductedsubstantially in accordance with the procedure described in A-V.

As tumor cells, human tumor cells (muscle fibrosarcoma HT-1080) wereused, and 5 days after the transplantation, drug administration wasstarted.

The sequence of drug administrations was as follows: A lipid emulsioncontaining the malate of formula (I) (the lipid emulsion of Example 2)was intravenously administered, and 20±5 minutes later, adriamycin (ADM)was intravenously administered. The administration was further performedevery 5 days from the day of starting the administration, and the totalnumber of administrations was therefore 4.

Mitomycin C (MMC) was tested by the same way as above.

Results

The doses of the drugs in the individual groups are shown in Table 10-1.

Thirty-six days after the transplantation of the tumor, a tumor mass wasextracted and its weight was measured. From the difference of the weightof the tumor from the non-treated control group, the tumor growthinhibition ratio (%) was calculated. The results are shown in Tables10-1 and 10-2.

                  TABLE 10-1                                                      ______________________________________                                                                            Tumor                                                                         growth in-                                                 Day of             hibition                                          Dose     adminis-   Tumor   ratio                                     Drug    (mg/kg)  tration    weight  (%)                                       ______________________________________                                        Control --       --         3.62 ± 1.78                                                                        --                                        ADM     4        5, 10, 15, 20                                                                            0.14 ± 0.32                                                                        96.1                                      Lipid   .sup.  5.sup.(1)                                                                       5, 10, 15, 20                                                emulsion                                                                      +                           0.03 ± 0.05                                                                        99.2                                      ADM     4        5, 10, 15, 20                                                ______________________________________                                         .sup.(1) Calculated as the malate of formula (I)                         

                  TABLE 10-2                                                      ______________________________________                                                                            Tumor                                                                         growth in-                                                 Day of             hibition                                          Dose     adminis-   Tumor   ratio                                     Drug    (mg/kg)  tration    weight  (%)                                       ______________________________________                                        Control --       --         3.62 ± 1.78                                                                        --                                        MMC     3        5, 10, 15, 20                                                                            1.92 ± 0.27                                                                        47.0                                      Lipid   .sup.  5.sup.(1)                                                                       5, 10, 15, 20                                                emulsion                                                                      +                           0.56 ± 0.28                                                                        84.5                                      MMC     3        5, 10, 15, 20                                                ______________________________________                                         .sup.(1) Calculated as the malate of formula (I)                         

It is seen from the results given in these tables that the lipidemulsion of this invention significantly potentiated the activities ofADM and MMC against HT-1080.

The results of the foregoing pharmacological tests show that the malateof formula (I) in accordance with this invention has excellentanti-cancer activity and excellent activity of potentiating theactivities of other anti-cancer agents, and that the lipid emulsioncontaining the malate of formula (I) has an excellent effect ofpotentiating the activities of other anti-cancer agents.

Now, a test for the stability of the lipid emulsion of this inventionwill be described.

[B] STABILITY TEST

The micro-emulsions containing theN-solanesyl-N,N,-bis(3,4-dimethoxybenzyl)ethylenediamine malate providedby this invention were tested for 3 months for stability. The contentwas measured by high-performance liquid chromatography (device: 655-15made by Hitachi Limited), and the particle size distribution analyzer(CAPA-500, made by Horiba Limited). The results are shown in Table 11.In a stability test at room temperature (25° C.) for 3 months, almost nochange was observed in content, appearance, pH and particle diameter.Accordingly, the micro-emulsion of this invention is very stablepharmaceutically.

                  TABLE 11                                                        ______________________________________                                        Stability Test                                                                               Period of observation                                                               Immediately                                              Micro-               after pre- 1      3                                      emulsion                                                                              Test items   paration   month  months                                 ______________________________________                                        Example 2                                                                             Content (mg/ml)                                                                            20.22      20.26  19.89                                          (residual rate, %)                                                                         (100.0)    (100.2)                                                                              (98.4)                                         Appearance   White non- --     --                                                          transparent                                                                   emulsion                                                         pH           3.59       3.60   3.62                                           Mean particle                                                                              0.21       0.21   0.21                                           diameter (μm)                                                      Example 3                                                                             Content (mg/ml)                                                                            20.28      20.36  19.93                                          (residual rate, %)                                                                         (100.0)    (100.4)                                                                              (98.3)                                         Appearance   White non- --     --                                                          transparent                                                                   emulsion                                                         pH           3.60       3.60   3.58                                           Mean particle                                                                              0.21       0.21   0.21                                           diameter (μm)                                                      Example 4                                                                             Content (mg/ml)                                                                            20.56      20.52  20.13                                          (residual rate, %)                                                                         (100.0)    (99.8) (97.9)                                         Appearance   White non- --     --                                                          transparent                                                                   emulsion                                                         pH           3.63       3.65   3.64                                           Mean particle                                                                              0.22       0.22   0.22                                           diameter (μm)                                                      Example 5                                                                             Content (mg/ml)                                                                            20.17      20.15  19.79                                          (residual rate, %)                                                                         (100.0)    (99.9) (98.1)                                         Appearance   White non- --     --                                                          transparent                                                                   emulsion                                                         pH           3.62       3.63   3.62                                           Mean particle                                                                              0.22       0.22   0.22                                           diameter (μm)                                                      Example 6                                                                             Content (mg/ml)                                                                            20.04      20.04  19.68                                          (residual rate, %)                                                                         (100.0)    (100.0)                                                                              (98.2)                                         Appearance   White non- --     --                                                          transparent                                                                   emulsion                                                         pH           3.65       3.65   3.63                                           Mean particle                                                                              0.20       0.20   0.20                                           diameter (μm)                                                      ______________________________________                                    

The invention will now be illustrated by the following examples showingthe production of the compound of formula (I) and the lipid emulsion ofthe invention.

EXAMPLE 1

Production of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediaminemalate of formula (I)

Twenty grams (0.02 mole) ofN-solaneyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine was dissolved in200 ml of ethanol, and a solution of 4.1 g (0.03 mole) of malic acid in10 ml of ethanol was added, and the mixed solution was gently heated.Ethanol was then evaporated under reduced pressure. The residue waswashed with ether, and recrystallized from a mixture of ethanol andether to give 18.2 g (yield 78.7%) of the captioned malate as colorlesscrystals having a melting point of 42° to 44° C.

Acute toxicity test

Using mice, the malate of formula (I) obtained as above was tested fortoxicity. It was found that in oral administration, no abnormality wasfound in the mice at a dose of 2,000 mg/kg. Toxicity was neither noted.

EXAMPLE 2

4.0 g of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malatewas added to 20 g of soybean oil described in the JapanesePharmacopoeia, and the mixture was dissolved under heat. Then, 2.4 g ofpurified soybean phospholipid and 5 g of glycerol were added to thesolution, and the mixture was vigorously stirred under heat. A suitableamount of distilled water was added, and the mixture was stirred by apolytron homogenizer to prepare a clude emulsion. The crude emulsion wasemulsified under high pressure by a Gaulin high-energy type homogenizer,and distilled water was added to adjust the amount of the emulsion to200 ml. There was obtained a micro-emulsion containingN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate. Thedispersed lipid particles had a mean particle diameter of 0.21 micron,and it did not contain particles having a size of at least 1 micron.

EXAMPLE 3

Example 2 was repeated except that 5 g of glycerol was used instead of13 g of D-sorbitol. A micro-emulsion containingN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate wasobtained.

EXAMPLE 4

4.0 g of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malatewas added to 20 g of soybean oil described in the JapanesePharmacopoeia, and the mixture was heated to form a solution. To thesolution were added 2.4 g of purified yolk phospholipid and a suitableamount of distilled water. The mixture was stirred by a polytronhomogenizer to prepare a crude emulsion. The crude emulsion wasemulsified under high pressure by a Gaulin high-energy homogenizer, anddistilled water was added to make 200 ml. A microemulsion containingN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate wasobtained. The dispersed lipid particles in the micro-emulsion had a meandiameter of 0.22 micron, and it did not contain particles having a sizeof at least 1 micron.

EXAMPLE 5

4.0 g of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malatewas added to 10 g of soybean oil described in the Japanese Pharmacopoeiaand 10 g of MCT, and dissolved under heat. Purified soybean phospholipid(1.2 g), 1.2 g of purified yolk phospholipid and 5 g of glycerol wereadded, and the mixture was vigorously stirred under heat. Afterdissolving, a suitable amount of water was added, and the mixture wasstirred by a polytron homogenizer to form a crude emulsion. The crudeemulsion was emulsified under high pressure by a Gaulin high-energyhomogenizer. Distilled water was added to make 200 ml. A micro-emulsioncontaining N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediaminemalate was obtained. The dispersed lipid particles in the micro-emulsionhad a mean diameter of 0.22 micron, and it did not contain particleshaving a size of at least 1 micron.

EXAMPLE 6

Example 2 was repeated except that 20 g of soybean oil was used insteadof 10 g of soybean oil. Thus, a micro-emulsion containingN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate wasobtained.

The dispersed lipid particles in the microemulsion had a mean particlediameter of 0.20 micron, and it did not contain particles having a sizeof at least 1 micron.

What is claimed is:
 1. A intravenously injectable oil-in-water typemicro-emulsion having an action of potentiating the activities ofanti-cancer agents, said micro-emulsion comprisingfine particles of amean diameter of not more than 1 micron of a vegetable oil or atriglyceride of a medium-chain fatty acid having 8 to 12 carbon atomscontaining 0.1 to 10% (w/v) ofN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate offormula (I) below, ##STR4## an aqueous medium, and
 0. 05 to 25% (w/v) ofphysiologically acceptable phospholipid for dispersing said fineparticles in said aqueous medium.
 2. The micro-emulsion of claim 1wherein the vegetable oil is pharmaceutically acceptable soybean oil. 3.The micro-emulsion of claim 1 wherein the physiologically acceptablephospholipid is a purified vegetable oil phospholipid.
 4. Themicro-emulsion of claim 3 wherein the purified vegetable oilphospholipid is purified soybean oil phospholipid.
 5. The micro-emulsionof claim 1 which consists essentially of5 to 50% (w/v) of fine particlesof a vegetable oil or a triglyceride of a medium-chain fatty acid having8 to 12 carbon atoms containing 0.1 to 10% (w/v) ofN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate,
 0. 05to 25% (w/v) of physiologically acceptable phospholipid,an isotonizingagent selected from the group consisting of glycerol, sugar alcohols,monosaccharides, disaccharides and amino acids in an amount sufficientto isotonize the emulsion, and water.
 6. The micro-emulsion of claim 1which consists of5 to 30% (w/v) of fine particles of soybean oil havingdissolved therein 0.3 to 3% (w/v) ofN-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine malate, 0.5 to25% (w/v) of purified soybean oil phospholipid, 1 to 5% (w/v) ofglycerol, and the remainder being water.
 7. The micro-emulsion of claim1 wherein the fine particles of the oil have a mean particle diameter ofnot more than 0.3 micron.
 8. The micro-emulsion of claim 4 wherein thepurified soybean oil phospholipid has a phosphatidyl choline content ofat least 80% by weight and an iodine value of 35 to 45.